JP5458565B2 - Method for producing succinate - Google Patents

Description

  The present invention relates to a method for producing succinate produced by fermentation of microorganisms. Specifically, the present invention relates to a method for producing succinate, which includes a step of obtaining a solution containing high-purity succinate from a non-permeate side by passing a microorganism culture solution through a nanofiltration membrane.

  Since succinic acid is a dicarboxylic acid in addition to uses such as food additives and pharmaceutical raw materials, it is widely applied to industrial uses as a monomer raw material for forming diol and polyester, and demand is increasing. Since this succinic acid can be produced by fermentation culture, it is also attracting attention as a polymer derived from non-petroleum raw materials. In general, succinic acid production by fermentation culture is carried out while maintaining an optimum pH for microorganisms by adding an alkaline substance to the culture solution. Therefore, a salt with the alkaline substance to be added is formed in the culture solution. In order to obtain high purity succinic acid, it is necessary to purify the succinate with high purity. The purified succinate is also used as a food additive. Sodium succinate is a umami ingredient, and calcium succinate and potassium succinate are contained in supplements and the like as nutritional supplement ingredients.

  As a method for obtaining such a succinate from a culture solution, there is a method of concentrating the culture solution under reduced pressure to evaporate water. However, succinate extracted from microbial fermentation broth contains impurities derived from medium components such as sugar and HMF (hydroxymethylfurfural) in addition to metabolites such as ethanol, glycerin and protein produced in the fermentation process. There are many. In addition, the purity of succinate is low, and heating is necessary to evaporate water, resulting in high energy costs.

Until now, as a method for producing an organic acid salt, a technique for treating an ultrafiltration membrane or a nanofiltration membrane in order to concentrate calcium lactate when producing a food containing calcium lactate has been disclosed. Adjusts the pH of a permeate obtained by treating a fermentation liquor containing calcium lactate with a filtration membrane to obtain calcium lactate. However, those skilled in the art will understand that permeate contains acetic acid in addition to lactic acid. Easily predicted. Therefore, even if this method is applied to the purification of succinate, the purity of the obtained succinate is not expected to be high (see Patent Document 1).
JP 2001-299281 A

  The present invention solves the above-described problem, that is, the problem of removing impurities in the culture solution when producing succinate by microbial fermentation, and efficiently recovers high-purity succinate. The purpose is to provide.

  As a result of intensive studies to solve the above problems, the inventors of the present invention have included succinate in the culture solution by passing the microbial fermentation culture solution adjusted to pH 6 to 9 through the nanofiltration membrane. The aqueous solution was recovered from the non-permeate side, and it was found that impurities could be removed from the permeate side with high efficiency, and the present invention was completed. That is, this invention is comprised from following (1)-(5).

  (1) A method for producing succinate by separating succinate produced in a culture solution by fermentation culture of microorganisms, wherein the culture solution having a pH adjusted to 6 or more and 9 or less is applied to a nanofiltration membrane. A method for producing succinate, comprising a step of collecting a succinate-containing solution from the non-permeate side.

  (2) The method for producing a succinate according to (1), wherein the functional layer of the nanofiltration membrane contains polyamide.

  (3) The method for producing a succinic acid salt according to (2), wherein the polyamide contains a crosslinked piperazine polyamide as a main component and contains a constituent represented by Chemical Formula 1.

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3).

  (4) The method for producing a succinate according to any one of (1) to (3), wherein the succinate is a sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt of succinic acid.

  (5) The succinic acid according to any one of (1) to (4), comprising a step of concentrating the succinic acid-containing solution obtained in the step with a reverse osmosis membrane and recrystallizing the succinate from the concentrated solution. Method for producing acid salt.

  The method for producing succinate according to the present invention can effectively remove impurities contained in a fermentation broth by a simple operation. Therefore, a high purity succinate can be obtained.

  Hereinafter, the present invention will be described in more detail.

  The method for producing succinate of the present invention is a method comprising a step of purifying succinate produced in a culture solution by fermentation culture of microorganisms, and adjusting the pH of the culture solution to 6 or more and 9 or less. Then, the culture solution is passed through a nanofiltration membrane to remove impurities in the culture solution to the permeate side, and an aqueous solution containing succinate is recovered from the non-permeate side.

  The microorganism used in the present invention is not particularly limited, and may be isolated from the natural environment. It may also be a microorganism whose properties have been partially modified by mutation or genetic recombination, for example, bacteria such as yeast, Escherichia coli and coryneform bacteria, filamentous fungi, actinomycetes or animals often used in the fermentation industry. Cells that are cultured cells such as cells or insect cells and modified so that succinate can be produced are also included in the microorganism used in the present invention. Specific examples of the succinate fermentation method include anaerobic spirolimus succinici producers (Anaerobiospirillum succinic products) (ATCC 35488) and an aerobic coryneform bacterium. There is known a method in which a genetically modified bacterium of Brevibacterium flavum is grown and then anaerobically acted on an organic raw material in a carbon dioxide-containing solution (Japanese Patent Laid-Open No. 11-196888).

  The fermentation raw material used for the fermentation culture of microorganisms may be any material that promotes the growth of the microorganisms to be cultured and can satisfactorily produce the desired fermentation product, succinate, carbon source, nitrogen source, inorganic A normal liquid medium containing salts and organic micronutrients such as amino acids and vitamins as appropriate is preferable. Carbon sources include sugars such as glucose, sucrose, fructose, galactose, lactose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, and organic acids such as acetic acid, alcohols such as ethanol And glycerin are also used. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, corn Steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added. When the microorganism used in the present invention requires a specific nutrient (for example, an amino acid) for growth, the nutrient is added as such or as a natural product containing it. Moreover, you may use an antifoamer as needed. In the present invention, the culture solution refers to a solution obtained as a result of the growth of microorganisms on the fermentation raw material. You may change suitably the composition of the fermentation raw material added to a culture solution from the fermentation raw material composition at the time of a culture | cultivation start so that the productivity of the target succinate may become high.

A succinate-containing culture solution produced by fermentation culture was obtained by adding an alkaline substance containing a monovalent or divalent cation to the culture solution and maintaining the pH optimum for microbial fermentation. A culture solution is preferably used. The alkaline substance to be added preferably contains a cation of sodium, potassium, magnesium, calcium and ammonium ions, more preferably a substance containing basic calcium and magnesium ions is added. Basic calcium includes calcium hydroxide, calcium carbonate, calcium phosphate, calcium oxide, calcium acetate, and basic magnesium includes magnesium hydroxide, magnesium chloride, magnesium carbonate, magnesium diphosphate, Examples include magnesium oxide. In the present invention, fermentation culture of microorganisms is preferably in the range of pH 4-8 and temperature 20-40 ° C. In addition, in the fermentation culture of microorganisms, if it is necessary to increase the oxygen supply rate, oxygen is added to the air to maintain the oxygen concentration at 21% or higher, or the culture is pressurized, the stirring speed is increased, the aeration rate is increased, etc. The following means can be used. In addition, anaerobic culture may be performed in order to increase the productivity of succinate during the growth of microorganisms and / or during fermentation production.

  In the step of separating succinate from the succinate-containing culture broth in the present invention, succinic acid has a pKa1 of 4.21 and a pKa2 of 5.64 at 25 ° C. In order to form a salt and efficiently separate and recover succinate on the non-permeate side of the nanofiltration membrane, it is necessary to adjust the succinate-containing culture solution to pH 6 or higher, preferably free succinic acid is used. It is pH 7 or higher that does not exist. In addition, when the pH of the succinate-containing culture solution is higher than 9, it affects the damage of the nanofiltration membrane, so it needs to be lower than that. The pH may be adjusted during fermentation culture or after completion of fermentation culture, but it is preferably performed after fermentation culture in consideration of succinate productivity of microorganisms.

  The nanofiltration membrane used in the method for producing succinate of the present invention is also called a nanofilter membrane (nanofiltration membrane, NF membrane), and “monovalent ions permeate and divalent ions pass through. It is a film generally defined as “blocking film”. It is a membrane that is considered to have a minute gap of about several nanometers, and is mainly used to block minute particles, molecules, ions, salts, and the like in water.

  Further, in the method for producing succinate of the present invention, “through a nanofiltration membrane” means that a succinate-containing culture solution is filtered through a nanofiltration membrane, and a succinate-containing solution is provided on the non-permeate side. It means to collect by filtration and allow impurities to permeate to the permeate side.

  The impurities contained in the succinate-containing culture solution in the present invention are not particularly limited as long as they are substances other than succinate contained in the culture solution, but in the present invention, they are byproducts of fermentation. Alcohols such as ethanol and glycerin are preferably removed as impurities from the permeate side. Further, hydroxymethylfurfural (HMF) and furfural known as impurities derived from the medium are also preferably removed from the permeate side.

  As a material for the nanofiltration membrane used in the present invention, a polymer material such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer can be used. It is not limited to this, and a film including a plurality of film materials may be used. In addition, the membrane structure has a dense layer on at least one side of the membrane, and on the asymmetric membrane having fine pores gradually increasing from the dense layer to the inside of the membrane or the other side, or on the dense layer of the asymmetric membrane. Either a composite film having a very thin functional layer formed of another material may be used. As the composite membrane, for example, as described in JP-A-62-201606, a composite membrane in which a nanofiltration membrane composed of a functional layer of polyamide is formed on a support membrane made of polysulfone as a membrane material can be used. .

  Among these, a composite film containing polyamide as a functional layer having high pressure resistance, high water permeability, and high solute removal performance and having an excellent potential is preferable. In order to maintain durability against operating pressure, high water permeability, and blocking performance, a structure in which polyamide is used as a functional layer and is held by a support made of a porous membrane or nonwoven fabric is suitable. As the polyamide semipermeable membrane, a composite nanofiltration membrane having a functional layer of a crosslinked polyamide obtained by polycondensation reaction of a polyfunctional amine and a polyfunctional acid halide on a support is suitable.

  In the nanofiltration membrane containing polyamide as a functional layer, preferred carboxylic acid components of monomers constituting the polyamide include, for example, trimesic acid, benzophenone tetracarboxylic acid, trimellitic acid, pyrometic acid, isophthalic acid, terephthalic acid, naphthalene Aromatic carboxylic acids such as dicarboxylic acid, diphenyl carboxylic acid, pyridine carboxylic acid and the like can be mentioned, but in view of solubility in a film forming solvent, trimesic acid, isophthalic acid, terephthalic acid or a mixture thereof is more preferable.

  Preferred amine components of the monomers constituting the polyamide include m-phenylenediamine, p-phenylenediamine, benzidine, methylenebisdianiline, 4,4′-diaminobiphenyl ether, dianisidine, 3,3 ′, 4- Triaminobiphenyl ether, 3,3 ′, 4,4′-tetraaminobiphenyl ether, 3,3′-dioxybenzidine, 1,8-naphthalenediamine, m (p) -monomethylphenylenediamine, 3,3′- Monomethylamino-4,4′-diaminobiphenyl ether, 4, N, N ′-(4-aminobenzoyl) -p (m) -phenylenediamine-2,2′-bis (4-aminophenylbenzimidazole), 2 , 2′-bis (4-aminophenylbenzoxazole), 2,2′-bis (4-aminophenyl) Secondary diamines such as primary diamines having an aromatic ring such as nylbenzothiazole), piperazine, piperidine or derivatives thereof, and the like. Among them, a nanofiltration membrane including a polyamide containing piperazine or piperidine as a monomer as a functional layer is pressure resistant. It is preferably used because of its heat resistance and chemical resistance in addition to its properties and durability. More preferably, the cross-linked piperazine polyamide or the cross-linked piperidine polyamide is a main component and a polyamide containing the constituent represented by the chemical formula 1, more preferably the cross-linked piperidine polyamide is a main component and the chemical formula 1 Polyamide containing the indicated constituents. In the above chemical formula 1, those having n = 3 are preferably used. Examples of the polyamide containing a crosslinked piperidine polyamide as a main component and containing the structural component represented by the chemical formula 1 include those described in JP-A No. 62-201606. As a specific example, a crosslinked piperazine polyamide is used. Examples thereof include UTC60, which is a crosslinked piperazine polyamide-based nanofiltration membrane having n = 3 in the chemical formula 1 as a main component.

  The nanofiltration membrane is generally used as a spiral membrane element, but the nanofiltration membrane used in the present invention can also be preferably used as a spiral membrane element. Specific examples of preferable nanofiltration membranes include, for example, GE Osmonics nanofiltration membrane GEsepa which is a cellulose acetate-based nanofiltration membrane, Alfa Laval nanofiltration membrane NF99 or NF99HF having a functional layer of polyamide. NF-45, NF-90, NF-200, or NF-400, a nanofiltration membrane manufactured by Filmtec Co., Ltd. having a cross-linked piperazine polyamide as a functional layer, and represented by the chemical formula (1) SU-210, SU-220, SU-600, and SU-610 are nanofiltration membrane modules manufactured by Toray Industries, Inc., which includes a polyamide-based nanofiltration membrane UTC60 manufactured by Toray Industries, Inc., which has a functional layer of a polyamide containing a constituent component. Alfa laval with polyamide as the functional layer NF99 or NF99HF of a nanofiltration membrane manufactured by NF-45, NF-90, NF-200 or NF-400 of a nanofiltration membrane manufactured by Filmtec Co., Ltd. having a functional layer of a crosslinked piperazine polyamide, or a crosslinked piperazine polyamide as a main component, And the nanofiltration membrane module SU-210, SU-220, SU-600 or SU-610 made by the company including UTC60 made by Toray Industries, Inc. having a functional layer of polyamide containing the constituent represented by the chemical formula (1) More preferably, the nanofiltration membrane module SU manufactured by the same company, including UTC60 manufactured by Toray Industries, Inc., which has a cross-linked piperazine polyamide as a main component and a polyamide containing a constituent represented by the chemical formula (1) as a functional layer. -210, SU-220, SU-600 or SU-610.

  In the succinate production method of the present invention, the filtration of the succinate-containing culture solution with the nanofiltration membrane may be performed under pressure. The filtration pressure is preferably used in the range of 0.1 MPa to 8 MPa. If the filtration pressure is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane may be damaged. Moreover, if the filtration pressure is 0.5 MPa or more and 7 MPa or less, the membrane permeation flux is high, so that the succinate-containing solution can be efficiently filtered, and there is little possibility of affecting the membrane damage. More preferably, it is more preferably used at 1 MPa or more and 6 MPa or less.

  As a method for evaluating the permeability of the nanofiltration membrane of succinate, it is difficult to measure the succinate concentration. Therefore, it can be evaluated by calculating the succinate permeability. A method of calculating the succinic acid concentration by high performance liquid chromatography is preferably used, but is not limited thereto. The succinic acid permeability is determined by analysis represented by high performance liquid chromatography. The succinic acid concentration (raw succinic acid concentration) contained in the raw water (culture solution) and the succinic acid concentration contained in the permeated water (succinic acid solution). By measuring (permeated water succinic acid concentration), it can be calculated by Equation 1.

  Succinic acid permeability (%) = (permeated water succinic acid concentration / raw water succinic acid concentration) × 100 (Equation 1).

  In addition, the transmittance of impurities can be calculated in the same manner as Equation 1.

  As the membrane separation performance of the nanofiltration membrane used in the succinate production method of the present invention, a sodium chloride aqueous solution (500 mg / L) adjusted to a temperature of 25 ° C. and a pH of 6.5 was evaluated at a filtration pressure of 0.75 MPa. Those having a salt removal rate of 45% or more are preferably used. The salt removal rate referred to here can be calculated by Equation 4 by measuring the permeated water salt concentration of the sodium chloride aqueous solution.

  Salt removal rate = 100 × {1− (salt concentration in permeated water / salt concentration in feed water)} (Formula 2).

In addition, the membrane permeation performance of the nanofiltration membrane is such that the membrane permeation flux (m ³ / (m ² · day)) is 0.5 or more at a filtration pressure of 0.3 MPa with sodium chloride (500 mg / L). Is preferably used.

  The concentration of succinate in the succinate-containing culture solution used for separation by the nanofiltration membrane is not particularly limited, but if it is high concentration, it is suitable for cost reduction because the concentration time can be shortened, For example, 10 g / L or more and 100 g / L or less is preferable.

  A solid succinate can be obtained by concentrating the nanofiltration membrane non-permeate and recrystallizing the succinate. As a concentration method, a method using a concentrator represented by an evaporator is general and can be applied in the present invention. However, since the heat capacity of water is much larger than that of an organic solvent, the energy and time required for concentration are enormous. It is. On the other hand, concentration using a reverse osmosis membrane is superior to concentration using an evaporator from the viewpoint of energy and cost reduction, and is preferably applied in the present invention.

  The reverse osmosis membrane in the present invention is a filtration membrane that removes ions and low molecular weight molecules using a pressure difference equal to or higher than the osmotic pressure of water to be treated as a driving force. For example, cellulose-based cellulose such as cellulose acetate and a polyfunctional amine compound A film in which a polyamide separation functional layer is provided on a microporous support film by polycondensation with a polyfunctional acid halide can be employed. In order to suppress fouling on the reverse osmosis membrane surface, the surface of the polyamide separation functional layer is coated with an aqueous solution of a compound having at least one reactive group that reacts with an acid halide group and remains on the surface of the separation functional layer. A low fouling reverse osmosis membrane mainly for sewage treatment in which a covalent bond is formed between the acid halogen group to be reacted and the reactive group can also be preferably employed. Since most of the divalent ions have been removed in the step of filtering through the nanofiltration membrane of the present invention, stable membrane concentration can be achieved without the formation of scale on the reverse osmosis membrane surface.

  Also, “through the reverse osmosis membrane” means that the succinate-containing solution passed through the nanofiltration membrane is concentrated through the reverse osmosis membrane, and the solution containing succinate on the concentrated liquid side is recovered. Means.

  As a reverse osmosis membrane preferably used in the present invention, a composite membrane using a cellulose acetate-based polymer as a functional layer (hereinafter also referred to as a cellulose acetate-based reverse osmosis membrane) or a composite membrane using a polyamide as a functional layer (hereinafter, Polyamide-based reverse osmosis membrane). Here, as the cellulose acetate-based polymer, organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and the like, or a mixture thereof and those using mixed esters can be mentioned. It is done. The polyamide includes a linear polymer or a crosslinked polymer having an aliphatic and / or aromatic diamine as a monomer. As the membrane form, an appropriate form such as a flat membrane type, a spiral type, and a hollow fiber type can be used.

  Specific examples of the reverse osmosis membrane used in the present invention include, for example, low pressure type SU-710, SU-720, SU-720F, SU-710L, SU, which are polyamide-based reverse osmosis membrane modules manufactured by Toray Industries, Inc. -720L, SU-720LF, SU-720R, SU-710P, SU-720P, high pressure type SU-810, SU-820, SU-820L, SU-820FA, SU-820FA, cellulose acetate System reverse osmosis membrane SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100, SC-8200, manufactured by Nitto Denko Corporation NTR-759HR, NTR-729HF, NTR-70SWC, ES10-D, ES20-D, ES20 U, ES15-D, ES15-U, LF10-D, Alfa Laval RO98pHt, RO99, HR98PP, CE4040C-30D, GE GE Sepa, Filmtec BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040, SW30HRLE-4040, etc. are mentioned.

  In the present invention, filtration of the nanofiltration membrane permeate with a reverse osmosis membrane is performed by applying pressure, but if the filtration pressure is lower than 1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane damage is affected. In order to give, it is preferable that it is the range of 1 MPa or more and 8 MPa or less. Moreover, if the filtration pressure is in the range of 1 MPa or more and 7 MPa or less, the membrane permeation flux is high, so that the succinate solution can be efficiently concentrated. Most preferably, it is in the range of 2 MPa or more and 6 MPa or less because there is little possibility of affecting the film damage.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to a following example.

(Reference Example 1) Evaluation of succinic acid permeability of nanofiltration membrane 100 g of succinic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to 10 L of ultrapure water to prepare a 10 g / L succinic acid aqueous solution. Next, calcium hydroxide was added and stirred at 25 ° C. for 1 hour to adjust the pH to 4, 6, 7, and 9, respectively. These aqueous solutions were each injected into the raw water layer 1 of the membrane filtration device shown in FIG. As a 90φ nanofiltration membrane indicated by reference numeral 7 in FIG. 2, a crosslinked piperazine polyamide semipermeable membrane “UTC60” (Nanofiltration membrane 1; manufactured by Toray), a crosslinked piperazine polyamide semipermeable membrane “NF-400” (nanofiltration membrane 2; Film Tech), polyamide semipermeable membrane “NF99” (nanofiltration membrane 3; manufactured by Alfa Laval), and cellulose acetate semipermeable membrane “GEsepa” (nanofiltration membrane 4; manufactured by GE Osmonics) are each made of stainless steel (manufactured by SUS316). It was set in a cell, the raw water temperature was adjusted to 25 ° C., the pressure of the high pressure pump 3 was adjusted to 0.5 MPa, and filtration was performed. The succinic acid concentration contained in the raw water tank 1 and the permeate 4 was analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the following conditions, and the succinic acid permeability was calculated.

  Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation), mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min), reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Vistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min), detection method: electrical conductivity, temperature: 45 ° C.

  The results are shown in Table 1.

  As shown in Table 1, even when any membrane was used, the higher the pH, the lower the succinic acid permeability. In addition, GEsepa (nanofiltration membrane 4) has low permeability at pH 4 and high permeability at pH 9 compared to other nanofiltration membranes, so cellulose acetate membranes have organic acids and succinates. It was suggested that the selectivity of was low.

(Example 1) Purification of calcium succinate from culture solution <Production of calcium succinate by microbial fermentation>
Succinate was produced by fermentation using the following method. 20 g / L glucose, 10 g / L polypeptone, 5 g / L yeast extract, 3 g / L K ₂ HPO ₄ , 1 g / L NaCl, 1 g / L (NH ₄ ) ₂ SO ₄ , 0.2 g / L 100 mL of a seed culture medium consisting of L MgCl ₂ and 0.2 g / L CaCl ₂ .2H ₂ O was placed in a 125 mL Erlenmeyer flask and sterilized by heating at 121 ° C. and 2 atm for 20 minutes. In an anaerobic glove box, 1 mL of 30 mM Na ₂ CO ₃ and 0.15 mL of 180 mM H ₂ SO ₄ were added, and further comprised of 0.25 g / L cysteine · HCl and 0.25 g / L Na ₂ S. After adding 0.5 mL of the reduced solution, Anaerobiospirillum succinici producers (CC) was inoculated with ATCC 53488, and statically cultured at 39 ° C. overnight to prepare a seed culture solution. Moreover, 1 L of fermentation medium shown in Table 2 was prepared in a mini jar fermenter (manufactured by ABLE, BMJ type, 2 L), and sterilized by heating at 120 ° C. and 2 atm for 20 minutes.

CO ₂ gas was passed through the sparger at 10 mL / min through 1 L of the fermentation medium, 10 mL of 3M Na ₂ CO ₃ was added, and the pH was adjusted to 6.8 with a sulfuric acid solution. Thereafter, 0.5 mL of a reducing solution consisting of 0.25 g / L cysteine / HCl and 0.25 g / L Na ₂ S is added, 50 mL of the seed culture solution is inoculated, and the culture is performed at a stirring speed of 200 rpm and 39 ° C. went. During the culture, pH was adjusted to 6.4 using 5M Ca (OH) ₂ . As a result of analysis by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the same conditions as in Reference Example 2, the amount of succinic acid accumulated in 39 hours of culture was 39 g, the production rate was 0.97 g / L / hr, and the production yield. Was 0.775 g / g. This culture solution was sterilized by heating at 120 ° C. for 20 minutes, centrifuged at 5000 × g for 20 minutes to remove the cells, and the supernatant was collected to obtain a calcium succinate-containing culture solution.

<Production of calcium succinate by addition of calcium hydroxide and NF membrane filtration>
Hydroxylation of calcium succinate-containing culture solution (2 L each) so that the pH becomes 6.8 (Examples 1, 3, 5, 7: pH unadjusted) and pH 9 (Examples 2, 4, 6, 8) After adding calcium (Wako Pure Chemical Industries, Ltd.), the mixture was stirred at 25 ° C. for 1 hour. Next, the calcium succinate-containing solution 2L was injected into the raw water tank 1 of the membrane filtration apparatus shown in FIG. As the 90φ nanofiltration membrane denoted by reference numeral 7 in FIG. 2, the nanofiltration membranes 1 to 4 are set in stainless steel (SUS316) cells, respectively, and the pressure of the high-pressure pump 3 is adjusted to 3 MPa, so that the permeate becomes 1 L. Nano filtration membrane filtration was performed until it became. The succinic acid and acetic acid concentrations contained in the concentrated water 5 and the permeate 4 under the respective conditions were analyzed by high performance liquid chromatography (manufactured by Shimadzu Corporation) under the same conditions as in Reference Example 1. Further, the HMF and furfural concentrations were measured by high performance liquid chromatography (Beckman colter) under the following conditions. The ethanol concentration was measured by gas chromatography (manufactured by Shimadzu Corporation). Table 3 shows the concentration of each component in the culture solution. Table 4 shows the results after purification of the nanofiltration membrane.

-Liquid chromatography measurement condition column: Synergies Hydro-RP (GL Science)
Mobile phase: water: acetonitrile = 95: 5
Detector: UV283nm
Gas chromatography measurement condition column: TC-1 0.53 mmI. D. × 15m df = 1.5um (GL Science)
Mobile phase: helium gas (7.9 mL / min, 50-100 ° C .: 5 ° C./min)
Detection: FID 250 ° C

  As a result of comparing the results shown in Tables 3 and 4, it was shown that the impurity content with respect to calcium succinate decreases because the impurities escape to the permeate side by NF membrane filtration.

(Example 2) Recrystallization of calcium succinate using reverse osmosis membrane 1 L of the succinic calcium-containing solution obtained in Example 1 was placed in the raw water tank 1 of the membrane filtration device shown in FIG. As a 90φ reverse osmosis membrane of No. 7, UTC70 (manufactured by Toray Industries, Inc.) was set in a cell made of stainless steel (manufactured by SUS316), the pressure of the high pressure pump 3 was adjusted to 5 MPa, and 0.9 L of permeated water was removed. This concentrated water 0.1L was left still at 4 degreeC overnight, and the crystal | crystallization of the calcium succinate was produced | generated. This was collected by suction filtration to obtain calcium succinate having a dry weight of 91.5 g. A saturated solution of this calcium succinate was prepared, and the concentrations of ethanol, HMF, furfural and acetic acid were measured by high performance liquid chromatography under the same conditions as in the above example. As a result, all impurities were below the detection limit (0.5 ppm). Therefore, it was shown that high-purity calcium succinate can be obtained by the present invention.

It is a schematic diagram showing one embodiment of the nanofiltration membrane and reverse osmosis membrane separation device used in the present invention. It is the schematic which shows one Embodiment of the cell sectional view with which the reverse osmosis membrane of the nanofiltration membrane and reverse osmosis membrane separation apparatus used by this invention was mounted | worn.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Raw water tank 2 Cell equipped with nanofiltration membrane or reverse osmosis membrane 3 High pressure pump 4 Flow of membrane permeate 5 Flow of membrane concentrate 6 Flow of culture solution or nanofiltration membrane permeate sent by high pressure pump 7 Nanofiltration membrane or reverse osmosis membrane 8 Support plate

Claims (5)

A method for producing succinate by separating succinate produced in a culture solution by fermentation culture of microorganisms, wherein the culture solution adjusted to a pH of 6 to 9 is passed through a nanofiltration membrane, A method for producing succinate, comprising a step of removing hydroxymethylfurfural (HMF) or furfural in the culture broth on the permeate side and recovering a succinate-containing solution from the non-permeate side.   The method for producing a succinate according to claim 1, wherein the functional layer of the nanofiltration membrane contains polyamide. The method for producing a succinate according to claim 2, wherein the polyamide comprises a crosslinked piperazine polyamide as a main component and a constituent represented by Chemical Formula 1.

(In the formula, R represents —H or —CH ₃ , and n represents an integer of 0 to 3.)   The method for producing a succinate according to any one of claims 1 to 3, wherein the succinate is a sodium salt, potassium salt, magnesium salt, calcium salt or ammonium salt of succinic acid. Concentration of the succinate salt-containing solution obtained by the process by the reverse osmosis membrane, comprising the step of recrystallizing the succinate salt from the concentrated solution, producing a succinate salt according to any one of claims 1 to 4 Method.

Images